Iron is an essential nutrient for nearly all organisms because it serves as a co-factor for many proteins, often in the form of iron, heme or iron-sulfur (Fe-S) clusters. Yet iron is often scarce in the environment placing its acquisition and usage under control of some of the most highly regulated pathways in biology. We are studying two key regulators of iron metabolism in the facultative anaerobe Escherichia coli, IscR and Fur. We have found that the activity and genes that are transcribed by IscR and Fur change significantly under anaerobic conditions. Thus, this project seeks to understand how this change occurs and how it impacts the utilization of iron under anaerobic conditions. Our first goal is to understand how activity of Fur, the global regulator of iron homeostasis in this and other bacteria, is regulated under anaerobic conditions. We will determine if an increase in free iron under anaerobic conditions can explain the increased activity of Fur at select weak affinity promoters in the absence of oxygen. We discovered that IscR is a novel DNA-binding protein that recognizes different sequence motifs in its apo- and [2Fe-2S]-bound forms. A second goal of this proposal is to define the biochemical and structural properties of IscR that allow metal binding to alter its DNA-binding specificity under anaerobic conditions. In this funding period, we will extend our studies of Fur and IscR from the well-studied K12 laboratory E. coli strain to the clinically relevant uropathogenic E. coli CFT073. Uropathogenic E.coli is a major cause of urinary tract infections and studies in model systems have linked both Fur and IscR to pathogenesis by this strain. Our results will provide important new insight as to how function of Fur and IscR are altered under anaerobic conditions. In addition, understanding how the activity of these regulators are coordinated to control iron and Fe-S homeostasis will allow us to develop new strategies to control the growth of commensal and pathogenic microbes in the anaerobic environment of the host.